key: cord-311811-nrodyagi
authors: Schutzer, Steven E.
title: The use of host factors in microbial forensics
date: 2019-12-06
journal: Microbial Forensics
DOI: 10.1016/b978-0-12-815379-6.00014-3
sha: 
doc_id: 311811
cord_uid: nrodyagi

Advances have been made in the forensic analysis of microbes and toxins. An underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. For forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. Principal methods that are sufficiently developed are characterization of the humoral immune response to microbial antigens including vaccine-induced immunity and detection of antibiotics that may be present in a possible perpetrator. This chapter presents central elements of the host response in a simplified fashion and describes a representative example, which, in the appropriate context, has a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim. This chapter also presents information about the immune system so that the interested reader can have a fuller understanding of the immune response in general.

Considerable advances have been made in the forensic analysis of microbes and toxins. These advances include sequencing, genomics, and microscopy. An underdeveloped and underutilized area in microbial forensics is how the host interacts with microorganisms in a way that provides unique signatures for forensic use. For forensic purposes, an immediate goal is to distinguish a potential victim and innocent person from a perpetrator, and to distinguish between a naturally acquired or intentional infection. Two principal methods that are sufficiently developed are characterization of the humoral immune response and identification of vaccine-induced immunity or antibiotics that may be present in a possible perpetrator.

This chapter presents central elements of the host response in a simplified fashion and describes a few representative examples, which, in the appropriate context, have a high potential of providing evidence that may aid an investigation to distinguish a perpetrator from a victim who has been exposed to a particular microbe or by-product, such as a toxin. This chapter also presents nonmicrobial forensicedirected information about the immune system so that the interested reader can have a fuller understanding of the immune response in general.

The primary aims of a microbial forensics are to identify the biological agent, its source, and the individuals responsible for the event (Budowle et al., 2003) . Analytic approaches differ when the suspected biothreat agent is encountered in a container or the environment, as opposed to in vivo in a human, animal, or plant. Analyses of trace elements, pollens, growth media, latent fingerprints, and microbial and nonmicrobial nucleic acids are all applicable to the container and environmental sample (States et al., 1999) . However, once the microorganism or its toxin is in the living host, it is no longer possible to analyze the preceding items except the microbial nucleic acid. However, the host's response to the biological agent may be available for analysis for clues. This is akin to other forensic studies where physical traces of bite marks, scratches, wound trajectories, and sizes of wounds are often surrogate evidence of the teeth, fingernails, and bullets (Averill and Odontology, 1991) . While the forensic pathologist is familiar with evidence related to determining the manner of death including the host response, those involved with healthcare alone are more familiar with the host response. In the context of microbial forensics, it is important to integrate all of these with intelligence information so that they may be included in the analytical data and attribution picture.

The physician and other healthcare providers may be among the first to realize that a patient is a victim of a biocrime. In the case of a covert attack, it may be the physician or medical examiner who first recognizes the index case. These healthcare workers are in key positions to preserve critical evidence and, thereby, contribute to the investigation (Schutzer et al., 2005) . There are a number of steps that should be followed when the possibility of a biological attack arises, either with the consent of the patient or because individuals are compelled by law to interact with public health and law enforcement.

A joint statement by the FBI, the CDC, and the DHS advises calling the FBI and public health authorities if a suspicious situation arises (Investigation et al.) . Some guidelines on the procedure(s) to report of suspicions of biocrimes are provided by the Centers for Disease Control and Prevention (CDC; http://www.cdc.gov), the Federal Bureau of Investigation (FBI; http://www.fbi.gov), and the Department of Homeland Security (DHS; http://www.dhs. gov) and detailed in previous article (Schutzer et al., 2005) .

The host response to a microorganism or other foreign substance is often a wellorchestrated series of events, which may protect the individual from harm (Zabriskie, 2009 ). At the same time, these host responses may provide clues as to identity of the offending microorganism or toxin as well as a rough chronology of when it occurred and for how long it has been persisting. Emerging technologies such as transcriptional arrays and bioinformatic analysis will eventually be refined and methods validated to provide even greater help in delineating more of the pathways and components of the host response to an infectious agent (Sala et al., 2009; Popper et al., 2009; Ko et al., 2015) . Other technologies are sufficiently mature to be of use today. The immune system and its components are a mainstay of our protection against infections and malignancies (Zabriskie, 2009; Paul, 2008; Murphy and Weaver, 2016) . Inflammation is often a side effect as the immune system contains and eradicates a microorganism or eliminates foreign tissue. Specific arms of the immune system can be used as markers in support of or against the presence of an infection. The humoral or antibody response to an invading microorganism is one example of a specific arm that can have forensic value. Some of the antibodies produced may have a protective role together with other parts of the immune system by eradicating the pathogen or neutralizing a toxin. Other antibodies may not be as effective in this role. However, in their ability to recognize unique and specific microbial antigens, they can serve as indicators that a specific microorganism was recently present or was present in the past. In the case of a vaccine, specific antibodies may recognize highly specific epitopes of one microbe versus those of a related microbe (e.g., influenza virus). This is especially so with different recombinant vaccines and could have forensic importance. Substances such as antibiotics, which can rapidly kill a pathogen, may modify the immune response by removing or reducing the infectious driving force for a full-scale response. As noted above, in clinical and veterinary medicine, measurement of the immune response helps the diagnostician decide what infection was present and how recently. In these situations, the intent is to provide treatment. The forensic scientist may exploit parts of the immune response to discover who is likely a victim of an attack and who might be responsible. This chapter will discuss the basics of the host immune response in a simplified manner that can have utility in a forensic sense. Examples will provide a sense of what information is achievable and what is 14. The use of host factors in microbial forensics not likely to provide clues with a high degree of certainty.

In response to a new exposure to a microbe, the innate immune system may be the first line of defense. Then the immune system starts to activate the antibody system. Typically, a cell known as a macrophage ingests and degrades some of the invading pathogens. It then presents part (antigens) of the microorganism to a helper T cell (a lymphocyte), which then directs other lymphocytes known as B cells to produce antibodies to those antigens of that particular microbe that were presented. It usually takes at least 4 days before any microbe-specific antibody can be detected (Parslow, 2001) .

Antibodies are a specific form of the proteins known as immunoglobulins (Igs). IgM, IgG, IgA, and secretory IgA are the principal classes of immunoglobulins with prime relevance to this chapter and will be discussed in more detail. In an infection, immunoglobulins usually appear in the order of IgM, IgG, and IgA. B cells first begin to produce IgM, and then some B cells undergo an irreversible switch to those that produce IgG. Later, some of these B cells undergo a switch to become IgA-secreting B cells. Immunoglobulins persist for varying times; for example, the half-life of particular IgM antibodies is approximately 5 days, while that of IgG can be as long as 21e23 days (Table 14 .1) (Paul, 2008; Murphy and Weaver, 2016) .

In certain circumstances of ruling in or ruling out a suspect, the specific IgE may be of value. Those individuals unfortunate to have allergies have problems due to IgE against allergens (such as ragweed, peanut, or cat dander). In this case, the IgE molecules sit on the surface of mast cells and basophils. These cells can release histamine and other allergic mediators when the offending allergen bridges two IgE molecules.

Similar to the effect from an infection with a live microbe, vaccines are often designed to provoke an antibody response. The vaccine can be composed of a live or attenuated microbe, a whole nonproliferating microbe, or an antigenic (recombinant) part of the microbe or a toxoid. Regardless, the intent of immunization with a vaccine is to engender protection, often by the generation of protective neutralizing antibodies. Although the half-life of an individual IgG molecule is less than a month, a population of antibodies of the IgG isotype form may persist for life. Memory B cells can sustain these antibodies and retain the ability to quickly generate the appropriate antibodies when challenged. When the immune system encounters another infection or is subjected to a revaccination (booster), the result is an accelerated production of the particular antibody and increase in the levels of antibodies that circulate in the blood ( Fig. 14.1) .

Perhaps, the pattern of antibody response which has the most forensic value, by providing a timeframe, is the appearance of IgM first, followed by a B-cell switch to the longer-lasting IgG. During the early phase of exposure, IgM predominates, as time goes on, IgG may wax and wane and IgM is no longer found ( Fig. 14.2) .

The antibody response to a particular agent may be directed to different antigens at different times, that is, early or later after the initial exposure. That response often involves IgM at the early stage and IgG later. Late in the course of (Bennett et al., 2015) , a virus known to cause mononucleosis. During acute early disease, it is common to find high levels of antibody of the IgM isotype to the viral early antigen (EA) and viral capsid antigen (VCA). It is rare to find IgG antibody to the VCA or to EpsteineBarr nuclear acid (EBNA) in anything but low titers (levels). As the patient recovers from their first infection with EBV, it is rare to find anything but low levels of IgM to EA or VCA, but IgG to VCA in higher or increasing levels is common. Antibodies to EBNA are often very low during this stage. Several months after clinical recovery, IgM to EA and VCA remain at low levels, whereas IgG to VCA and EBNA are present at high levels, often for years. 

A controlled experiment or normal clinical event illustrates what happens when the immune system responds to the infectious agent or a vaccine again. The controlled experiment may be in a laboratory animal or a patient Illustrative concepts receiving a booster vaccine. The uncontrolled but normal clinical event occurs when the patient is reexposed to the infectious agent. Consider a generic antigen exposure. The first time the immune system encounters antigen X (AgX), it responds as shown in Figs. 14.1 and 14.2. Initially, antibodies to AgX are barely discernible; then levels rise and later fall to a plateau. If a simultaneous exposure were to occur with AgX and a new AgY from another microorganism, the immune system would quickly mount a brisk response with high levels of Ab to AgX, while the course of Ab to AgY would be slow and delayed, just as it was in the response to the first exposure to AgX. This phenomenon is termed immunological memory or an amnestic response. This can be useful when the symptoms and signs of exposure to either X or Y are similar. This is the case with the early flu-like symptoms of pulmonary anthrax (Raymond et al., 2009; Waterer and Robertson, 2009; Bush et al., 2001) and with the influenza virus itself (Meltzer et al., 2010; Cao et al., 2009; Lessler et al., 2009) .

Another example common to all of us is repetitive exposure to different strains of flu viruses (Meltzer et al., 2010; Janeway, 2001) . As illustrated in Table 14 .4, a person infected for the first time with one strain of the influenza virus has a response to most of its antigens (as a theoretical example, Ag 1, 2, 3, 4, 5, 6). Three years later, the same individual exposed to a partially similar influenza virus responds preferentially to those antigens that were also present on the original influenza virus. The person also makes a smaller initial antibody response to new antigens, that is, those not shared with the first virus. Ten or 20 years later, during a new flu season and exposure to a third strain of influenza, the most brisk responses would be to antigens previously recognized by the immune system. This is the scientific basis for giving the flu vaccine, which contains a variety of possible antigens common to multiple strains of the flu virus so that a rapid and protective antibody response will occur. Utility of serologic analysis of people exposed to anthrax: strengths and limitations

Our knowledge of the humoral response to infection with biothreat microbes is limited compared with our knowledge of the kinetic response to common human infections. Nevertheless, in the appropriate context and with sufficient background information, detection of antibodies to a particular microbe and its antigens can have important value for a microbial forensic investigation. This information may have critical probative value or it can guide investigative leads. The absence of a specific antibody response may also have value in a particular investigation. Certainly, its importance is increased in the context of information of what organism could be involved, when the exposure was likely to have occurred, the route of exposure, what symptoms and signs are manifesting in the host, and other data such as presence of antigens and microbial nucleic acids (Jackson et al., 1998) . Other information such as how many hosts (people or animals) have had this infection in the geographic region, what is the incidence, and background prevalence of antibody titer to the organism in question or a related organism, in the population being studied, is also important.

Vaccination responses can have forensic value. The current protective antigen (PA) vaccine has small amounts of EF and lethal factor (LF), which are responsible for some of the side effects, so one might expect to see antibodies against these antigens as well as to PA. The recombinant PA is just PA so anti-LF and anti-EF would be absent in an immunized individual.

The 2001 anthrax-letter attacks raised multiple questions for every person infected, possibly exposed, vaccinated, or treated. Some of these questions included how these persons were infected by spores, if at all; that is, through breaks in the skin (cutaneous anthrax); by inhalation of spores (pulmonary anthrax (Bennett et al., 2015) ), or by ingestion (gastrointestinal anthrax (Bravata et al., 2007; Tutrone et al., 2002) ). Or, were they among the "worried well"?

Consider the situation where a close associate comes down with symptoms compatible with inhalational anthrax after receiving a letter containing powder and that material is no longer available. Until this is shown not to be anthrax, great worry will ensue.

In several cases of documented exposure, there was not enough time for the patient to develop antibody to a specific anthrax antigen, at least as probed for IgG. Serial serum samples obtained on November 16, 17, 18, and 19 of 2001 were tested for IgG antibody to the PA component of the anthrax toxins by enzymelinked immunosorbent assay (ELISA); all samples were nonreactive. Serial tests for serum IgG antibody to the PA toxin of anthrax were performed on 436 workplace-exposed persons. All but one test was negative. Most of the specimens were collected on October 10 and 17 (Traeger et al., 2002) .

It is instructive to look at the positive antibody case in the context of the nature and Utility of serologic analysis of people exposed to anthrax: strengths and limitations duration of that individual's symptoms when he developed a positive test. Ernesto Blanco, a 73-year-old mailroom clerk (case 2), experienced fatigue on September 24. He worked in the mailroom of the AMI building and delivered mail to the index case. On September 28, he developed a nonproductive cough, intermittent fever, runny nose, and conjunctivitis. These signs worsened through October 1 when he was hospitalized. In addition, he had shortness of breath with exertion, sweats, mild abdominal pain and vomiting, and episodes of confusion. His temperature was elevated to 38.5 C (101.3 F), heart rate was rapid at 109/min, respiratory rate was slightly fast at 20/min, and blood pressure was 108/61 mm Hg. He had bilateral conjunctival injection and bilateral pulmonary rhonchi. At the time of admission, his neurologic exam was normal.

No skin lesions were observed. The only laboratory abnormalities were low albumin, elevated liver transaminases, borderline low serum sodium, increased creatinine, and low oxygen content in the blood. Blood cultures were negative on hospital day 2, after antibiotics had been started. The chest X-ray showed a leftsided pneumonia and a small left pleural effusion but no "classical" mediastinal widening (Dewan et al., 2002) . The patient was initially given intravenous azithromycin; cefotaxime and ciprofloxacin were subsequently added. A nasal swab obtained on October 5 grew Bacillus anthracis on culture. Computed tomography (CT) of the chest showed bilateral effusions and multilobar pulmonary consolidation but still no significant mediastinal lymphadenopathy. Pleural fluid aspiration was positive for B. anthracis DNA by PCR. Bacterial cultures of bronchial washings and pleural fluid were negative. Immunohistochemical staining of a transbronchial biopsy demonstrated the presence of B. anthracis capsule and cell wall antigens. During hospitalization, his white blood count rose to 26,800/mm 3 , and fluid from a second thoracentesis was positive for B. anthracis DNA by PCR. Immunohistochemical staining of both pleural fluid cells and pleural biopsy tissue demonstrated the presence of B. anthracis capsule and cell wall antigens. Serial serum samples demonstrated > fourfold rise in serum IgG antibody to the PA component of the anthrax toxins by an ELISA assay. The patient was able to leave the hospital on October 23 on oral ciprofloxacin. Table 14 .5 illustrates both the clinical and microbial forensic approach and context in which to analyze such a patient. It is likely to be common to most situations where a biocrime (2003). These questions include was the infection acquired naturally or was it an intentional action that led to the infection; how did the particular individual acquire it if it was not a natural infectiondwas he the target or a bystander. An alternative possibility in the right circumstances is a laboratory-acquired infection. This case also demonstrates that cultures may be negative at different times from different fluids and tissues because of early administration of antibiotics. However, the remnants of the infection, even dead organisms, can be found by probing for antigens and DNA. This patient's response demonstrated a classic principle of infectious disease, a rising antibody titer over time. In this case, it was IgG to a particular antigenic component of the anthrax toxins (Friedlander and Little, 2009; Cunningham et al., 2002) . The subject's antibody response may have been detected earlier if IgM to this component or to other antigens of anthrax had been sought. The case also points out the utility of integrating the presence of antibody with other indications of an anthrax infection such as culture, PCR, and antigen detection. These take on their greatest significance during clinical illness in someone who was possibly exposed.

Early administration of antibiotics can prevent or interfere with the isolation of a pathogen by culture (Kaeberlein et al., 2002) . Of the first 10 pulmonary anthrax cases associated with the 2001 letter attacks, three patients had no isolate of B. anthracis from any clinical samples; however, culture was attempted after initiation of antibiotic therapy. History of exposure in conjunction with compatible symptoms and signs of disease and objective laboratory findings were the basis for the diagnosis. B. anthracis was identified in pleural fluid, pleural biopsy, or transbronchial biopsy specimens by reactivity with B. anthracis-specific cell wall and capsular antibodies or by the detection of DNA in pleural fluid or blood by PCR (Jernigan et al., 2001) .

It is important to understand the limitations of any assay used in medicine or forensics (Budowle et al., 2008; Schutzer et al., 2009 ). An IgG-based ELISA for anti-PA illustrates the importance of understanding the limitations of an assay. The ELISA was developed at the US Army Medical Research Institute of Infectious Disease (USAMRIID) and put into operation after optimization and internal validation at the CDC for functional sensitivity and specificity in detecting an antibody response to B. anthracis infection. Its major limitation was that only one antigen was used and only IgG was measured. Therefore, a negative result shortly after exposure may, in effect, be a false-negative result. A gap such as this may be filled by development of an assay for antigenspecific IgM, and by probing for other B. anthracis antigens or epitopes yet to be characterized.

The assay may be very useful in its present form to screen asymptomatic people with possible exposure. The study by Dewan et al. (Dewan et al., 2002) provide a contemporary background database on a group of postal workers who may have been exposed to B. anthracis. Beginning on October 29, 2001, 1657 postal employees and others who had been to the Washington D.C. postal facility went to the D.C. General Hospital for antibiotics in addition to those people whose treatment began on October 21, 2001. Serum samples were also obtained from the 202 individuals who had been to the Washington D.C. postal facility during the precious 2 weeks. All were negative for specific anti-PA IgG, including three individuals who reported a remote history of anthrax vaccination. The consistent negative findings may be explained by the fact that antibiotic therapy was initiated before serum testing and that there were no baseline serum samples available for testing. In addition, the time period from exposure to sampling was very short. Among 28 individuals in the Capitol region with culture-positive nasal swabs who received prophylactic antibiotics immediately, none had a positive culture from a nasal swab repeated 7 days later, and none developed IgG to PA antigen 42 days after exposure. This again emphasizes the limitation and interpretation of a test in someone who had early antibiotic treatment. It does raise forensic utility considerations. Even with these easily disseminated spores, an antibody response may be aborted or modified with antibiotics by early eradication. Furthermore, antibiotics taken before exposure would likely be effective in preventing laboratory and clinical signs of an infection. Detection of microbial DNA, antigen, or the organism itself on a person's body, clothing, or possessions should raise a red flag for exposure.

The route of infection is important in interpreting results and the limitations of the assay used. The example of cutaneous anthrax in Paraguay illustrates this notion, as well as the need to search for other antigens as markers of exposure (Harrison et al., 1989) . In an analysis of an outbreak of 21 cases of cutaneous anthrax that followed contact with raw meat from a sick cow, sera from 12 cases and 16 colony and 2 noncolony controls were examined by Western blot for antibodies to PA and LF 6 weeks after the outbreak. An ELISA was used to probe for antibodies to the poly-D-glutamic acid capsule. Of the 12 cases, 11 had antibody to PA, for a sensitivity of 91.7%; none of the 18 controls was positive. Only 6 of 12 cases had antibody to LF; all controls were negative. Anticapsule antibodies were positive in 11 of 12 but were also positive in 2 of 18 controls. The results of this study demonstrate the need to consider other antigens.

Some of the principles discussed above are highlighted by a report on severe acute respiratory syndrome (SARS). The appearance alone of this coronavirus responsible for this disease evoked concern of a possible terrorist origin at the onset. A report in the Morbidity and Mortality Weekly Report (MMWR (CfDCaP, 2003) ) on the "Prevalence of IgG Antibody to SARS-Associated Coronavirus in Animal Traders" discussed the need to validate and interpret tests in appropriate populations. Also discussed was the inability to date the time of infection by the IgG assay, and the possibility of assay crossreactivity to a near neighbor that might be unknown. In a Promed bulletin, Dr. Steve Berger looked at the same data from a different perspective and reported "This week's study in MMWR indicates that animal contact may indeed promote infection; however, the most striking finding seems to have eluded the authors: 1.2 percent to 2.9 percent of individuals in a healthy control group of adults were also found to be seropositive! The population of Guangdong Province is 86.42 million (2001), of whom 61.14 million are adults over age 14. If we assume that the seropositivity rates among controls is representative of the province as a whole, 734,000 to 1,773,000 adults in Guangdong have at some time been infected by the SARS virus. These figures are 87-to 211-fold the total number (8422) of SARS patients reported worldwide to date!" This comparison is a good illustration of the advantage of open dissemination and discussion of information as well as the need to question the methodology of acquisition of data before accepting their application in formulas or for analyses for forensics and epidemiology. It is also of value to remember that many infections include many with SARS coronavirus have been asymptomatic or mildly symptomatic.

Plague, is a zoonotic infection caused by Yersinia pestis, which occurs in the western United States with regularity and has an animal reservoir (Bennett et al., 2015) . The situation with the naturally occurring Yersinia is in contrast to the appearance of a case of smallpox which would raise an immediate red flag for a bioterrorist event. Cases need to be approached from an epidemiologic standpoint first to determine whether it is a naturally acquired case or whether the facts point to a deliberate introduction of the organism. Analytic techniques could include genomic analysis of an isolated organism and immunological response of the host. In the new era of rapid and deep sequencing, our capacity to investigate the genomics is growing (Mardis, 2008; Stavnsbjerg et al., 2017) . In consideration of animal reservoirs, ELISA assays were compared with other tests for detection of plague antibody and antigen in multimammate mice (Mastomys coucha and. Mastomys natalensis) (Shepherd et al., 1986) , which were experimentally infected and then sacrificed at daily intervals. IgG ELISA was equivalent in sensitivity to passive hemagglutination and more sensitive than the IgM ELISA and complement fixation. Antibody was detectable by Day 6 after infection using all four tests. IgM ELISA titers fell to undetectable levels after 8 weeks. Plague fraction 1 antigen was detected in 16 of 34 bacteremic sera from M. coucha and M. natalensis. This antibody pattern comparison shows that the principle of IgM versus IgG to this pathogen works to temporally situate the infection as an early versus late or past event. It also shows that when the information is combined with antigen detection, it engenders more confidence in the results. It should be noted that conclusions from this older reference has been substantiated with more defined antigens and assay technologies.

Melioidosis is caused by Burkholderia pseudomallei (Ashdown, 1992) . Key clinical signs and laboratory results may raise the possibility of an infection with this pathogen. Whether it is an acute, persistent, or past infection can be determined by assessing several host responses. Often a simple indicator such as erythrocyte sedimentation rate or C-reactive protein (CRP) can raise a clinical suspicion of an infection. In a study of 46 patients with clinical melioidosis, 35 (22 culture-positive and 13 culturenegative) had relatively uneventful disease courses. Initially, they had elevated serum CRP that decreased with antibiotic therapy and returned to normal as the disease resolved. In another series of patients, IgM and IgG were measured by ELISA in 95 sera from 66 septicemic cases and 47 sera from 20 cases with localized melioidosis (Chenthamarakshan et al., 2001) . Sixty-five sera from culture-negative cases seronegative for other endemic infections but suspected of melioidosis were also examined. Other controls included serum from 260 nonmelioidosis cases, 169 high-exposure risk cases, and 48 healthy individuals. The IgG-ELISA was 96% sensitive and 94% specific. All sera from cases with septicemic and localized infections and 61 of 63 sera from clinically suspected melioidosis cases were positive for IgG antibody. The sensitivity and specificity of the IgM ELISA were 74% and 99%, respectively. A geometric antibody index for IgM antibody in the sera of the melioidosis cases was significantly higher in cases compared with that of the noncase controls. In another study by some of the same authors, a rapid test for IgG and IgM was shown to have clinical utility (Cuzzubbo et al., 2000) . A study with the intent of evaluating the utility of an IgG assay compared with other assays illustrates how the clinical and temporal context must be integrated for interpretation (Dharakul et al., 1997) . It also illustrates how there is room for technical improvement in tests but the best setting is often the endemic area itself or at least using samples from that area in which the infections are occurring. These tests were evaluated in the actual clinical setting in an area endemic for melioidosis. Specificity of IgG (82.5%) and IgM (81.8%) assays was significantly better than that of an indirect hemagglutination test (IHA) (74.7%). The sensitivity of the IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the IgM test (63.5%). Specific IgG was found in septicemic cases (87.8%) and localized infections (82.6%). The IgG test was also better than the IgM test and the IHA test in identifying acute melioidosis cases in the first 5 days after admission. IgG antibody to a B. pseudomallei antigen remained high for longer than 5 years in recovered, disease-free patients. Because this is a disease that may have an incubation of days to years, an acute case may very well be detected by a rise in specific IgM if it were a matter of days from infection. Although endemic for Southeast Asia, if B. pseudomallei was used as a biothreat agent in a different environment, its course and manifestations may not be recognized due to unfamiliarity with the disease.

The above example also points out how the context in which a test is used determine is valuable. The concept of predictive value is instructive in determining how useful a test may be. In terms of disease detection, a high positive predictive value indicates the test is useful in determining that the disease is present. A high negativity predictive value would indicate that the test is useful in excluding the presence of the disease. Another zoonotic agent is Rift Valley fever virus (RVFV), which can be transmitted via aerosols (Clark et al., 2018) . One study with the intent at looking for improved tests showed the utility of IgM to determine an early exposure to RVFV (Niklasson et al., 1984) . Two ELISA IgM tests detected specific IgM antibodies to RVFV during the first 6 weeks after vaccination. Three inactivated vaccine doses were given on days 0, 6 to 8, and 32 to 34. ELISA serum IgM on days 6e8 were negative or in the lower range of detection; on days 32e34 the serum antibody values were strongly positive; on days 42e52, they were waning and in later collected samples were negative. The plaque reduction neutralization test was negative on days 6e8 and became positive in later samples. Similar to the examples shown above, these data suggest that three doses of RVFV vaccine induced a prolonged primary antibody response. The authors of that study concluded that the ELISA IgM may be useful for early diagnosis of acute human infection. Good correlation of a neutralization test and ELISA IgG would indicate a later infection.

Taken together, these examples illustrate that an ideal test or analysis for both clinical and forensic use would incorporate endemic and incident area controls, historical contextual information, knowledge of the route of exposure, background incidence, and kinetics of transmission.

Each of these scenarios must take into account multiple factors and the limitations of any analytic process to be applied. On one extreme is the situation that occurred with the onset of acquired immunodeficiency syndrome (AIDS) from the human immune deficiency virus (HIV) in the United States. Initially, there were no cases, and therefore a precise highly sensitive and specific test with excellent positive and negative predictive values (such as exists now when a combination of tests are used) would not likely yield a positive result in an area where there was little HIV infection and disease at the onset such as Kansas. A positive test by today's methodologies from a 1970 serum sample from Kansas would be considered a probable falsepositive and warrant further investigation. Today, several viral and nucleic acid assays are available that would provide a definitive diagnosis in a short period of time (Bennett et al., 2015) . However the same sample tested at the beginning of HIV testing could have been positive if the person had adult T-cell leukemia, which is caused by human T-cell leukemia virus-1 (HTLV-1) because the original tests for what became known as AIDS involved whole viral lysates in which up to 30% of the HTLV-1 sera cross-reacted. Questions regarding the interpretation of the test results could be raised by knowledge of different presentations of the infection. For example, HTLV-1 can actually be used in the laboratory to immortalize cells. In the patient, it actually increases the T-cell count, as is the nature with leukemia, instead of decreasing them, as with HIV infections. Other laboratory indicators such as hypercalcemia would now raise the leukemia as a consideration.

Interpretation of a positive laboratory test must also take into account the health status of the person being tested. This is important for the practice of medicine and can have relevance when extended to forensic analysis (Schutzer et al., 2005) . The following examples illustrate this concept. Individuals who have syphilis, a treponemal bacterial infection, can typically have a positive fluorescent treponemal antibody test result for years, even after successful treatment. However, while infected they would have a positive venereal disease research laboratory (VDRL) test, which reverts to negative following successful antibiotic therapy. The VDRL test detects nonspecific anticardiolipid antibodies and can produce false-positive results with other conditions (e.g., pregnancy). There are some notable exceptions related to crossreactive epitopes or autoimmune diseases. These are readily distinguishable by history and clinical information. Similarly, individuals infected with active tuberculosis will likely have a positive skin test (Mantoux) or a positive interferon-gamma release assay (Dewan et al., 2006; Ota and Kato, 2017) , whereas the uninfected healthy person will be negative. In certain instances, a sick person with a cell-mediated immune deficiency will be anergic, that is, he/she will be negative to multiple skin tests including common antigens such as Candida. The key difference here is that there is a great difference between the healthy person being tested and an ill or immunocompromised individual being subjected to the same test.

Tests may also discriminate between the length of the infection (i.e., acute or chronic); limitations of these tests may lead to different interpretations unless one is familiar with those limitations. An example of this occurred with the bacterial infection of Borrelia burgdorferi, which causes Lyme disease. Antibiotics can abrogate the antibody response because ELISA results were negative in 30% of patients with known disease who were treated early (Dattwyler et al., 1988) . In early cases, reactivity to a unique antigen, OspA, was also negative in serological assays despite a demonstrable T-cell Possible scenarios of bioterrorism attacks: distinguishing victims from perpetrators response (Krause et al., 1992) . Analysis of these same sera found that there was antibody to B. burgdorferi, but it was below the threshold of detection by conventional assays. It was detectable in its bound form, in immune complexes (Schutzer et al., 1990; Schutzer and Coyle, 2016) .

Anthrax can be used as an example where investigatory leads can be generated by considering a scenario in toto. The elderly woman who died in Connecticut from inhalation of anthrax clearly had no occupational exposure nor was she known to have had contact with anyone who had anthrax. It was possible that she had contact with cross-contaminated mail. However, if this case had occurred as the index case or out of context of the mail attacks, it would have been reasonable to question her travel history, what her work if any was, or if she received or used spore-contaminated products from an anthrax endemic area. Similarly, the Vietnamese woman who died of inhalation anthrax in New York City would also have had these questions investigated. It would have been useful to search for direct or indirect evidence of anthrax by physical examinations of her contacts or close neighbors. Inspection and cultures from her workplace, apartment, and apartment complex (especially contiguous neighbors) are important for detecting the presence of B. anthracis. Coworkers, friends, neighbors, and other contacts could have had their serum analyzed for antibody to antigens of B. anthracis. These samples could have been frozen so that if one were positive it would be available for a comparison study in the future. At a minimum, these types of studies could serve as future control data for the geographic region. With molecular methods, even trace amounts might be detectable (Lasken and Egholm, 2003) although parallel investigation as to background control would be necessary. Although hypothetical, several results could have occurred, and each will be considered separately. First example, a close contact is positive for IgM to one of the B. anthracis antigens, such as PA.

This finding would suggest that this person had recent exposure and, if nothing else, should be treated. This individual could conceivably be the one who knowingly or unknowingly passed the spores to the patient. Given the October 26 onset of illness, which is late in the mailing sequence, it would be less likely that this individual was a perpetrator but rather a recent victim. However, if this person were IgG-positive, then there are several other possibilities. Perhaps, this person had past exposure in an endemic region and was treated (e.g., Haiti, where anthrax is known as "charcoal disease"). Or this person could have been vaccinated for bona fide reasons such as a researcher who received it for occupational exposure. Or this person could have obtained the vaccine originally for legitimate or illegal purposes but was nevertheless vaccinated. The vaccine usage may have been for a clinical trial or animal experimentation. Animal vaccines may be more obtainable without strict record keeping. This person could have loaded the mail with relative impunity if there was protective antibody generated from the vaccination. Situations similar to this one will require intelligence information regarding access, ability, and motive. In an area where recombinant vaccines are being developed or used antibody response would be different between someone using one type of recombinant vaccine as compared with someone using another type of vaccine. Nevertheless, finding IgG to one or more antigens of B. anthracis could point investigators toward such a seropositive individual, whereas an IgM finding could justify critical therapy. Where information points to a particular individual, investigation could be extended to search for ingestion or injection of antibiotics as illustrated below in the ciprofloxacin example. Questions would be raised regarding access to antibiotics, recent ingestion/injection of them, half-life of the antibiotic, half-life of the metabolites of the antibiotics, and in which body fluids or tissues can the residual be found. As illustrated from 14. The use of host factors in microbial forensics the data in the earlier sections, someone with antibiotics in their system may be protected following exposure to a potential pathogen. This person would be antibody-negative and likely antigen-and microbial DNA/RNAnegative, because the infection would have been eradicated before the organism can proliferate in any significant quantity. The widespread prophylactic use of ciprofloxacin during the period following the anthrax mailing attacks is illustrative of an understudied area. Ciprofloxacin has been increasingly associated with tendonitis and ruptured Achilles tendons (Akali and Niranjan, 2008; Palin and Gough, 2006; Godoy-Santos et al., 2018) . In the future, better methodology to follow the pharmacokinetics of an antiinfective compound may have forensic implications. In the last example, someone who takes an antibiotic prophylactically while manipulating a lethal microbe may exhibit side effects that in the proper context of an investigation may add to the picture of possible culpability. This area is far from established at this point in time.

Strategies can be employed to examine suspicious but possible accidental transmission of infections. This approach is illustrated by a recent study of avian influenza using a multitude of assays. Tools to determine person-to-person spread as the mode of transmission included viral culture, serologic analysis, immunohistochemical assay, reverse transcriptasee polymerase chain reaction (RT-PCR) analysis, and genetic sequencing (Ungchusak et al., 2005; Meinel et al., 2018) .

It is likely that future understanding of the immune system and evolving technologies will bring new analytic power to the field, but in the interim we can make good use of proven principles for forensic purposes.

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